WO2000024390A1 - Procede et composition pour la modulation de l'amylose - Google Patents

Procede et composition pour la modulation de l'amylose Download PDF

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Publication number
WO2000024390A1
WO2000024390A1 PCT/US1999/023885 US9923885W WO0024390A1 WO 2000024390 A1 WO2000024390 A1 WO 2000024390A1 US 9923885 W US9923885 W US 9923885W WO 0024390 A1 WO0024390 A1 WO 0024390A1
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abc
blocker
abc transporter
amyloid
transporter
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PCT/US1999/023885
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English (en)
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WO2000024390A9 (fr
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Peter B. Reiner
Fred Chiu-Lai Lam
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The University Of British Columbia
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Priority to EP99954894A priority Critical patent/EP1123090A1/fr
Priority to JP2000578000A priority patent/JP2002528411A/ja
Priority to CA002348019A priority patent/CA2348019A1/fr
Priority to IL14272599A priority patent/IL142725A0/xx
Priority to AU11128/00A priority patent/AU762593B2/en
Publication of WO2000024390A1 publication Critical patent/WO2000024390A1/fr
Publication of WO2000024390A9 publication Critical patent/WO2000024390A9/fr

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Definitions

  • Alzheimer's disease is a common dementing brain disorder ofthe elderly.
  • the key features ofthe disease include progressive memory impairment, loss of language and visuospatial skills, and behavior deficits. These changes in cognitive function are the result of degeneration of neurons in the cerebral cortex, hippocampus, basal forebrain, and other regions ofthe brain.
  • Neuropathological analyses of postmortem Alzheimer's diseased brains consistently reveal the presence of large numbers of neurofibrillary tangles in degenerated neurons and neuritic plaques in the extracellular space and in the walls ofthe cerebral micro vasculature.
  • the neurofibrillary tangles are composed of bundles of paired helical filaments containing hyperphosphorylated tau protein (Lee, V. M and Trojanowski, J.
  • the disordered Cytoskeleton in Alzheimer's disease Curr. Opin. Neurobiol. 2:653 (1992)).
  • the neuritic plaques consist of deposits of proteinaceous material surrounding an amyloid core (Selkoe, D. J., "Normal and abnormal biology of the ⁇ -amyloid. precursor protein", Annu. Rev. Neurosci. 17:489-517 (1994)).
  • amyloid- ⁇ peptide plays a significant role in the etiology of Alzheimer's disease.
  • a portion of this evidence is based upon studies which have been generated from data with regard to familial Alzheimer's disease. To date, this aggressive form of Alzheimer's disease has been shown to be caused by missense mutations in (at least) three genes: the amyloid precursor protein (APP) gene itself (Goate, A. et al., "Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease", Nature 349:704-706 (1991) and Mullan, M.
  • APP amyloid precursor protein
  • missense mutations in APP are located in the region ofthe protein where proteolytic cleavage normally occurs (see below), and expression of at least some of these mutants results in increased production of A ⁇ (Citron, M. et al., "Mutation ofthe ⁇ -amyloid precursor protein in familial Alzheimer's disease increases ⁇ -amyloid production", Nature 360:672-674 (1992), Cai, X-D. et al, "Release of excess amyloid ⁇ protein from a mutant amyloid ⁇ protein precursor", Science 259:514-516 (1993) and Reaume, A. G.
  • Alzheimer's disease apolipoprotein E is highly correlated with expression of Alzheimer's disease (Poirier, J., "Apolipoprotein E in animal models of CNS injury and in Alzheimer's disease", Trends Neurosci. 17:525-530 (1994); Roses, A. D.
  • a ⁇ deposited in sporadic Alzheimer's disease plaques is typically a longer 42 amino acid version, A ⁇ 42 (Gravina, S. A. et al., "Amyloid beta protein (A beta) in Alzheimer's disease brain: Biochemical and immunocytochemical analysis with antibodies specific for forms ending at A beta 40 or A beta 42", J. Biol. Chem. 270:7013-7016 (1995)).
  • a ⁇ 42 is particularly pathogenic is consistent with data which demonstrate that this isoform is more lipophilic, aggregates more easily, and is more neurotoxic than its 40 amino acid cousin A ⁇ 40 (Yankner, B. A., "Mechanisms of neuronal degeneration in Alzheimer's disease", Neuron 16:921-932 (1996)).
  • the A ⁇ 42 phenotypic similarity gives support to the hypothesis that sporadic Alzheimer's disease is also due to increased production of A ⁇ . What remains unclear is what the nature ofthe change is in sporadic Alzheimer's diseased brains which lead to increased production of A ⁇ .
  • a ⁇ deposition is an early and invariant event in Alzheimer's disease, it is believed that treatment which reduces production of A ⁇ will be useful in the treatment of this disease.
  • amyloidosis is also implicated in the pathophysiology of both stroke and head trauma.
  • cerebral amyloid angiopathy is a common feature ofthe brains of stroke patients with symptoms of dementia, focal neurological syndromes, or other signs of brain damage (Corio and Rubio, "Cerebral amyloid angiopathies", Neuropath Appl Neurohiol 22:216-227
  • APP is an ubiquitous transmembrane glycoprotein (Selkoe, D. j., "Normal and abnormal biology ofthe ⁇ -amyloid precursor protein", Annu. Rev. Neurosci. 17:489-517 (1994)).
  • Three major isoforms of APP are produced by alternative splicing: 751 and 770 amino acid isoforms contain a Kunitz protease inhibitor domain and are expressed both in neuronal and non-neuronal cells, while a 695 amino acid isoform lacks this domain and is expressed at high levels in neurons.
  • Mature APP is turned over rapidly, with a half life of -20-30 minutes.
  • Embedded within the protein is a sequence of 39-43 amino acids which corresponds to the A ⁇ peptide.
  • Proteolytic processing of APP yields peptide fragments of varying size.
  • An extensively studied degradative pathway is one in which the APP molecule is cleaved within the A ⁇ sequence (between residues 16 and 17) by a yet-to-be identified enzyme termed ⁇ -secretase.
  • the resultant ⁇ 110-125 kDa soluble extracellular derivative termed APP S is rapidly released into the extracellular medium of cultured cells (Weidemann, A. et al.. "Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein", Cell 57:115-126 (1989); Esch, F. S.
  • the A ⁇ peptide is secreted by cells (Haass, C. et al. "Amyloid ⁇ -peptide is produced by cultured cells during normal metabolism", Nature 359:322-325 (1992); Seubert, P. et al, “Isolation and quantitation of soluble Alzheimer ⁇ -peptide from biological fluids", Nature 359:325-357 (1992); Shoji, M. et al, "Production ofthe Alzheimer amyloid ⁇ protein by normal and proteolytic processing", Science 258:126- 129 (1992); Busciglio, J. et al., “Generation of ⁇ -amyloid in the secretory pathway in neuronal and nonneuronal cells", Proc. Natl.
  • a ⁇ may accumulate intracellularly and thereby initiate the disease process (Wild-Bode et al., "Intracellular generation and accumulation of amyloid ⁇ -peptide terminating at amino acid 42.” J. Biol. Chem. 272:16085-16088 (1997)). As a result, considerable effort is underway to unravel the molecular pathways mediating A ⁇ secretion.
  • a ⁇ can be generated both via a classical secretory pathway (Dyrks, T. et al, "Amyloid precursor protein secretion and ⁇ A4 amyloid generation are not mutually exclusive", FEBS Lett. 349:210-214 (1994); Busciglio, J. et al, "Generation of ⁇ -amyloid in the secretory pathway in neuronal and nonneuronal cells", Proc. Natl. Acad. Sci. USA 90:2092-2096 (1993); Citron, M. et al, "Generation of amyloid ⁇ protein from its precursor is sequence specific", Neuron 14:662-670 (1995); Perez, R. G.
  • APP S secretion is increased (Buxbaum, J. D. et al, "Processing of Alzheimer ⁇ /A4 amyloid precursor protein: Modulation by agents that regulate protein phosphorylation", Proc. Natl. Acad. Sci. USA 87:6003-6006 (1990);Caporaso, G. L. et al, "Protein phosphorylation regulates secretion of Alzheimer ⁇ /A4 amyloid precursor protein", Proc. Natl. Acad. Sci. USA 89:3055-3059 (1992); Slack, B. E.
  • MAP kinase (Mills et al., "Regulation of amyloid precursor protein catabolism involves the mitogen-activated protein kinase signal transduction pathway," J Neurosci 17:9415-9422 (1997); Desdouits-Magnen et al., “Regulation of secretion of Alzheimer amyloid precursor protein by the mitogen-activated protein kinase cascade," J Neurochem 70:524-530 (1998)), as does activation of nerve growth factor receptors (Schubert, D. et al, "The regulation of amyloid ⁇ protein precursor secretion and its modulatory role in cell adhesion", Neuron 3:689-694 (1989); Fukuyama, R.
  • Alzheimer's disease, stroke, e.g., cerebral ischemia, and head injury are characterized by cognitive and neurological deficits associated with extracellular and cerebrovascular amyloid deposits.
  • This invention provides methods, compositions, and screening methods which are useful in the treatment of amyloidosis.
  • the methods ofthe invention involve administering to a subject a pharmaceutical composition including one or more agents which modulate (e.g., inhibit, prevent, or enhance) production and/or release of A ⁇ and ultimately, amyloid deposition.
  • the methods and compositions ofthe invention are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs, e.g., Alzheimer's Disease, stroke and/or head trauma.
  • the methods ofthe invention can be used therapeutically to treat amyloidosis or can be used prophylactically in a subject susceptible to amyloidosis.
  • the methods ofthe invention are based, at least in part, on modulating cleavage of amyloid precursor protein (APP), the proteolytic processing of APP and/or exportation of amyloid- ⁇ protein (A ⁇ ) by a blocker of a member ofthe ATP binding cassette (ABC) superfamily of transporters expressed in the brain or the cerebral microvasculature, e.g., MDR1, MDR3, ABC1, ABC2, ABC3, ABC7, ABC8, MRP4, MRP5 or the human ABC transporters encoded by the ESTs 45597, 122234, 123147, 131042, 157481, 182763, 352188 or 422562.
  • APP amyloid precursor protein
  • a ⁇ amyloid- ⁇ protein
  • ABSC ATP binding cassette
  • a therapeutic agent such as a blocker, used in the method ofthe invention can modulate amyloid deposition.
  • the present invention provides methods for modulating amyloid deposition in a subject, by administering to the subject an effective amount of an ATP binding cassette (ABC) transporter or flippase blocker.
  • the modulation includes preventing or inhibiting the amyloid deposition.
  • the methods provide that one or more ABC transporter blockers or flippase blockers act to antagonize transport of A ⁇ through one or more ABC transporters or flippase blockers expressed in the brain or the cerebral microvasulature.
  • the present invention also provides methods for treating a disease state associated with amyloid deposition by administering to a subject an effective amount of at least one ABC transporter or flippase blocker, or a pharmaceutically acceptable salt thereof, such that a disease state associated with amyloidosis is treated.
  • the amyloid deposition is associated with Alzheimer's Disease.
  • the present invention also provides methods for treating Alzheimer's disease by administering to a subject having Alzheimer's disease an effective amount of at least one ABC transporter or flippase blocker, or a pharmaceutically acceptable salt thereof, such that treatment occurs.
  • the present invention pertains to methods for treating head trauma by administering to a subject having head trauma an effective amount of at least one ABC transporter or flippase blocker, or a pharmaceutically acceptable salt thereof, such that treatment occurs.
  • the present invention also pertains to methods for treating stroke by administering to a subject affected by a stroke an effective amount of at least one ABC transporter or flippase blocker, or a pharmaceutically acceptable salt thereof, such that treatment occurs.
  • the present invention further pertains to packaged pharmaceutical compositions for treating amyloidosis.
  • a packaged pharmaceutical composition includes a container which holds an effective amount of a pharmaceutical composition for modulating amyloid deposition in a subject.
  • the pharmaceutical composition includes at least one ABC transporter or flippase blocker and instructions for using the pharmaceutical composition.
  • the packaged pharmaceutical composition is for treatment associated with Alzheimer's Disease.
  • the present invention also pertains to methods for identifying agents which modulate amyloid deposition in an organism by administering to an organism an effective amount of at least one ATP binding cassette (ABC) transporter or flippase blocker, such that modulation of amyloid deposition occurs.
  • the methods provide that one or more ABC transporter blockers or flippase blockers which act to antagonize transport of A ⁇ through one or more ABC transporters or flippase blockers expressed in the brain or the cerebral microvasulature can be identified.
  • the present invention further pertains to methods for identifying agents which modulate transport or flipping of amyloid across a membrane, e.g., a cellular or synthetic membrane.
  • the methods include introducing an agent into a model system containing the membrane, e.g., cellular or planar lipid membrane, ABC transporter and amyloid. The ability ofthe agent to modulate the transport or flipping of amyloid across the membrane is measured.
  • the methods provide the ability to identify of one or more ABC transporter blockers or flippase blockers which act to antagonize transport of A ⁇ through one or more ABC transporters or ABC transporters having flippase activity which are expressed in the brain or the cerebral microvasulature.
  • FIG. 1 is a western blot of an SDS-PAGE gel demonstrating that RU-486 increases release of APP S from PC12 cells.
  • FIG. 2 is a graph comparing the effects of RU-486 and nerve growth factors upon APP S release from PC 12 cells.
  • FIG. 3 is a western blot of an SDS-PAGE gel demonstrating that RU-486 increases release of APP S from PC 12 cells in a dose-dependent manner.
  • FIG. 4 is a graph showing the dose-dependent effects of RU-486 administration upon APP S release from PC 12 cells.
  • FIG. 5 is a western blot of an SDS-PAGE gel demonstrating that RU-486 increases release of APP S from PC 12 cells after an exposure of only 15 minutes.
  • FIG. 6 is a western blot of an SDS-PAGE gel demonstrating that RU-486 increases release of APP S from E-82 cells which lack progesterone and glucocorticoid receptors.
  • FIG. 7 is a western blot of a SDS-PAGE gel demonstrating that RU-486,
  • Progesterone and Desmethoxyverapamil increase APP S secretion in PC 12 cells.
  • FIG. 8 is a graph of relative increase of APP S in Fig. 7.
  • FIG. 9 is a western blot of an SDS-PAGE gel demonstrating that RU-486 does not increase release of APP S from KB-3.1 cells which have minimal expression of p- glycoprotein.
  • FIG. 10 is a western blot of an SDS-PAGE gel demonstrating that transfection of KB-3.1 cells with an expression vector for the human p-glycoprotein gene restores regulation of APP S release by RU-486.
  • FIG. 11 is a western blot of a SDS-PAGE gel demonstrating that RU-486 decreases the release of A ⁇ from SW cells.
  • FIG. 12 is a western blot of an SDS-PAGE gel demonstrating that RU-49953 decreases the release of A ⁇ from SW cells.
  • FIG. 13 demonstrates transient transfection of P-glycoprotein increases basal secretion of ⁇ -amyloid.
  • FIG. 14 is a graphic depiction of densitometric analysis of transfected K269sw cells.
  • FIG. 15 illustrates results obtained from an exemplary in vitro screening assay for identifying agents which modulate transport of A ⁇ across membranes by measuring A ⁇ transport across the membrane, as set forth in further detail in XI ofthe Exemplification, below.
  • the present invention pertains to methods for modulating amyloid deposition in a subject by administering to the subject an effective amount of at least one transport or flippase blocker for an ATP binding cassette (ABC) transporter which is expressed in the brain or cerebral microvasculature, such that modulation of amyloid deposition occurs.
  • AAC ATP binding cassette
  • Amyloid deposition is a common problem associated with Alzheimer's disease, stroke and/or head trauma and is typically characterized by neuritic plaques.
  • the present invention also pertains to methods for treating a disease state associated with amyloidosis by administering to a subject an effective amount of at least one ABC transporter blocker, or a pharmaceutically acceptable salt thereof, such that a disease state associated with amyloidosis is treated.
  • the methods provide that one or more ABC transporter blockers or flippase blockers act to antagonize transport of A ⁇ through one or more ABC transporters or flippase blockers expressed in the brain or the cerebral microvasulature.
  • ABSC ATP binding cassette
  • ESTs expressed-sequence tags
  • human ABC transporters include MDR1 and the cystic fibrosis transmembrane regulator, although examples of ABC transporters are also well-known in other species such as the chloroquine transporter of Plasmodium falciparum and the yeast transporter ste-6. Ofthe 33 putative human ABC transporters, evidence suggests that at least 16 may be expressed in the brain or its microvasculature. Examples of human ABC transporters which are expressed in the brain and its microvasculature include MDR1, MDR3, ABC1, ABC2, ABC3, ABC7, ABC8, MRP4, MRP5, and the human ABC transporters encoded by the ESTs 45597, 122234, 123147, 131042, 157481, 182763, 352188 and 422562.
  • ABSC transporter is further intended to include a superfamily of genes found in many organisms including humans (Higgins CF, "ABC transporters: from microorganisms to man", Annu Rev Cell Biol 8:67-113 (1992)) characterized by a highly conserved region known as the ATP binding cassette (Hyde et al., "Structural model of ATP -binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport", Nature 346:362-365 (1990); Mimura et al, "Structural model ofthe nucleotide binding conserved component of periplasmic permeases", Proc Natl Acad Sci USA 88:84-88 (1991)).
  • the EST database can further be utilized as a first-pass means of examining expression of putative ABC transporters insofar as the expression library from which the EST derives can be used to provide partial information regarding the expression ofthe full-length gene. For example, an EST which is derived from an infant brain library is likely to be expressed in the brain.
  • the term "p-glycoprotein" is art recognized and is intended to include a subset of the family of ABC transporters.
  • P-glycoproteins are large, glycosylated membrane proteins that can function as ATP-dependent efflux pumps, and are encoded by two genes in humans known as MDR1 and MDR3 and three genes in the mouse known as mdrl, mdrl, and mdr3 (Endicott, JA and Ling, V, , "The biochemistry of p- glycoprotein-mediated multidrug resistance", Ann Rev Biochem 58:137-171 (1989)).
  • MDR1 is art recognized and is intended to include the human MDR1 encoded gene product (GenBank Accession No. M14758; Chen et al., "Internal duplication and homology with bacterial transport proteins in the mdrl (p-glycoprotein) gene from multidrug resistant human cells," Cell 47:381-389 (1986)) as well as its various isoforms as well as analogs, homologues, and orthologs in other species.
  • the MDR1 gene product is an integral membrane protein having a predicted molecular weight of approximately 170 kDa, and has been shown to act as an ATP-dependent efflux pump with wide substrate specificity (Endicott, JA and Ling, V, , "The biochemistry of p-glycoprotein-mediated multidrug resistance", Ann Rev Biochem 58:137-171 (1989)).
  • the human MDR1 gene is expressed in the cerebral microvasculature and astrocytic foot processes (Thiebaut et al., "Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein pi 70: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein.” J Histochem Cytochem 37:159-164 (1989); Pardridge et al., "Brain microvascular and astrocyte localization of p-glycoprotein,” J Neurochem 68:1278-1285 (1997)).
  • MDR3 is art recognized and is intended to include the human MDR3 encoded gene product (GenBank Accession No. M23234; Chen et al., "Internal duplication and homology with bacterial transport proteins in the mdrl (p-glycoprotein) gene from multidrug resistant human cells," Cell 47:381-389 (1986)) as well as its various isoforms as well as analogs, homologues, and orthologs in other species.
  • the MDR3 gene product is an integral membrane protein and has been shown to promote translocation of phosphatidylcholine (Smith et al., "The human MDR3 p-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice" FEBS Lett 354:263-266 (1994)).
  • a BLAST Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990) search of GenBank predicts that human MDR3 is encoded by ESTs corresponding to gb- AA677416, gb-AA456377, gb-AA459824, gb-W22853, and gb-R53330 (best 5 matches listed only). Because at least one of these ESTs derives from an infant brain library, MDR3 is predicted to be expressed in brain.
  • ABCV is art recognized and is intended to include the human ABC1 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • the mouse homologue of ABC1 has been cloned (EMBL Accession No.
  • the human gene encoding ABC1 has been partially cloned, is found on chromosome 9, and is expressed in brain (Luciani et al., "Cloning of two novel ABC transporters mapping on human chromosome 9," Genomics 21:150-159 (1994)).
  • a BLAST Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990) search of GenBank predicts that human ABC1 is encoded by ESTs corresponding to gb-U18236, gb- N46182, gb-H45142, gb-U66691, and gb-H21585 (best 5 matches listed only).
  • ABC2 is art recognized and is intended to include the human ABC2 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • the mouse homologue of ABC2 has been cloned (EMBL Accession No.X75927, Luciani et al., "Cloning of two novel ABC transporters mapping on human chromosome 9," Genomics 21:150-159 (1994)).
  • the human gene encoding ABC2 has been partially cloned, is found on chromosome 9, and is expressed in brain (Luciani et al., "Cloning of two novel ABC transporters mapping on human chromosome 9," Genomics 21:150-159 (1994)).
  • a BLAST Altschul et al., "Basic local alignment search tool;' J Mol Biol 215:403-410 (1990) search of GenBank predicts that human ABC2 is encoded by ESTs corresponding to gb-H39045, gb-U18235, gb-T33919, gb-W69928, and gb-M78056 (best 5 matches listed only).
  • ABSC3 is art recognized and is intended to include the human ABC3 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • the cDNA for human ABC3 (GenBank Accession No. U78735, Connors et al., "The cloning of a human ABC gene (ABC3) mapping to chromosome 16pl3.3,” Genomics 39:231-234 (1997)) is 5112 nucleotides in length, encoding 1704 amino acids with a predicted molecular weight of 191 kDa, is found on chromosome 16pl3.3, and is expressed in brain.
  • ABC7 is art recognized and is intended to include the human ABC7 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • the mouse homologue of ABC7 (abc7) has been partially cloned and is expressed in the brain (GenBank Accession No. U43892, Savary et al. "Isolation and chromosomal mapping of a novel ATP-binding cassette transporter conserved in mouse and human," Genomics 41 : 275-278 (1997)).
  • a BLAST Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990) search of GenBank predicts that human ABC7 is encoded by ESTs corresponding to gb-U66679, gb-AA403130, gb- AA626765, gb-AA668992, gb-AA733151 (best 5 matches listed only).
  • v45CS is art recognized and is intended to include the human ABC8 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • the gene encoding human ABC8 has been cloned (GenBank Accession No. U34919, Croop et al., "Isolation and characterization of a mammalian homologue of the Drosophila white gene," Gene 185:77-85 (1997); GenBank Accession No. X91249, Chen et al., “Cloning ofthe cDNA for a human homologue ofthe Drosophila white gene and mapping to chromosome 21q22.3," Am J Human Genetics.
  • a BLAST Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990) search of GenBank predicts that human ABC8 is encoded by ESTs corresponding to gb- AA297078, gb-AA297109, gb-AA305082, gb-AA297995, and gb-AA297906 (best 5 matches listed only).
  • MRP4 is art recognized and is intended to include the human MRP4 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • the human gene encoding MRP 4 has been partially cloned, is found on chromosome 13 (GenBank Accession No U83660, Kool et al., "Analysis of expression of cMOAT (MRP 2), MRP 3, MRP 4 and MRP 5, homologues ofthe multidrug resistance- associate protein gene (MRP1), in human cancer cell lines," Cancer Res 57:3537-3547 (1997)).
  • a BLAST Altschul et al, "Basic local alignment search tool," J Mol Biol 215:403-410 (1990) search of GenBank predicts that human MRP 4 is encoded by ESTs gb-R35797, gb-U66686, gb-R35798, gb-N66654, and gb-AA015868 (best 5 matches listed only). Because at least one of these ESTs derives from an infant brain library, MRP4 is predicted to be expressed in brain.
  • MRP5 is art recognized and is intended to include the human MRP5 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • the human gene encoding MRP5 has been partially cloned, is found on chromosome 3, and is expressed in brain (GenBank Accession No. U83661, Kool et al., "Analysis of expression of cMOAT (MRP2), MRP 3, MRP 4 and MRP 5, homologues of the multidrug resistance-associate protein gene (MRP1), in human cancer cell lines," Cancer Res 57:3537-3547 (1997)).
  • GenBank A BLAST (Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990)) search of GenBank predicts that human MRP 5 is encoded by ESTs gb-U66687, gb-H17207, gb-AA829904, gb-H60893, gb- R34891 (best 5 matches listed only).
  • human ABC transporter encoded by EST 45597 is art recognized and is intended to include the human EST 45597 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • EST 45597 was identified via BLAST (Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990)) search ofthe public EST database using the N-terminal ATP-binding domain of MDR1 as a conserved region of superfamily of ABC transporters (Allikmets et al., "Characterization ofthe human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database", Human Mol Genetics 5:1649-1655 (1996)). Because EST 45597 is found in an infant brain library, the human ABC transporter encoded by EST 45597 is predicted to be expressed in the brain.
  • human ABC transporter encoded by EST 122234 is art recognized and is intended to include the human EST 122234 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • EST 122234 was identified via BLAST (Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990)) search of the public EST database using the N-terminal ATP- binding domain of MDR1 as a conserved region of superfamily of ABC transporters (Allikmets et al., "Characterization ofthe human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database", Human Mol Genetics 5:1649-1655 (1996)). Because EST 122234 is found in an infant brain library, the human ABC transporter encoded by EST 122234 is predicted to be expressed in the brain.
  • human ABC transporter encoded by EST 123147 is art recognized and is intended to include the human EST 123147 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • EST 123147 was identified via BLAST (Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990)) search ofthe public EST database using the N-terminal ATP- binding domain of MDR1 as a conserved region of superfamily of ABC transporters (Allikmets et al., "Characterization ofthe human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database", Human Mol Genetics 5:1649-1655 (1996)).
  • human ABC transporter encoded by EST 123147 is predicted to be expressed in the brain.
  • the term "human ABC transporter encoded by EST 131042" is art recognized and is intended to include the human EST 131042 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • EST 131042 was identified via BLAST (Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990)) search ofthe public EST database using the N-terminal ATP- binding domain of MDR1 as a conserved region of superfamily of ABC transporters (Allikmets et al., "Characterization ofthe human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database", Human Mol Genetics 5:1649-1655 (1996)). Because EST 131042 is found in an infant brain library, the human ABC transporter encoded by EST 131042 is predicted to be expressed in the brain.
  • human ABC transporter encoded by EST 157481 is art recognized and is intended to include the human EST 45597 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • EST 157481 was identified via BLAST (Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990)) search ofthe public EST database using the N-terminal ATP- binding domain of MDR1 as a conserved region of superfamily of ABC transporters (Allikmets et al., "Characterization ofthe human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database", Human Mol Genetics 5:1649-1655 (1996)). Because EST 157481 is found in an infant brain library, the human ABC transporter encoded by EST 157481 is predicted to be expressed in the brain.
  • human ABC transporter encoded by EST 182763 is art recognized and is intended to include the human EST 182763 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • EST 182763 was identified via BLAST (Altschul et al, "Basic local alignment search tool," J Mol Biol 215:403-410 (1990)) search ofthe public EST database using the N-terminal ATP- binding domain of MDR1 as a conserved region of superfamily of ABC transporters (Allikmets et al, "Characterization ofthe human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database", Human Mol Genetics 5:1649-1655 (1996)). Because EST 182763 is found in an infant brain library, the human ABC transporter encoded by EST 182763 is predicted to be expressed in the brain.
  • human ABC transporter encoded by EST 352188 is art recognized and is intended to include the human EST 352188 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • EST 352188 was identified via BLAST (Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990)) search ofthe public EST database using the N-terminal ATP- binding domain of MDR1 as a conserved region of superfamily of ABC transporters (Allikmets et al., "Characterization ofthe human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database", Human Mol Genetics 5:1649-1655 (1996)). Because EST 352188 is found in an infant brain library, the human ABC transporter encoded by EST 352188 is predicted to be expressed in the brain.
  • human ABC transporter encoded by EST 422562 is art recognized and is intended to include the human EST 422562 gene product and its various isoforms as well as analogs, homologues, and orthologs in other species.
  • EST 422562 was identified via BLAST (Altschul et al., "Basic local alignment search tool," J Mol Biol 215:403-410 (1990)) search ofthe public EST database using the N-terminal ATP- binding domain o ⁇ MDRl as a conserved region of superfamily of ABC transporters (Allikmets et al., "Characterization ofthe human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database", Human Mol Genetics 5:1649-1655 (1996)).
  • EST 422562 is reported to be a housekeeping gene (Allikmets et al., "Characterization ofthe human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database", Human Mol Genetics 5:1649-1655 (1996)), the human ABC transporter encoded by EST 422562 is predicted to be expressed in the brain.
  • transporter blocker is intended to include those compounds that can modulate ABC transporters expressed in the brain or the cerebral microvasculature, such as those described supra, including MDR1, MDR3, ABC1, ABC2, ABC3, ABC7, ABC8, MRP4, MRP5 and the human ABC transporters encoded by the ESTs 45597, 122234, 123147, 131042, 157481, 182763, 352188 and 422562.
  • modulate includes effect(s) on ABC transporters that prevent or inhibit amyloid production and/or release, which may ultimately affect amyloid deposition, e.g., in the context ofthe therapeutic methods ofthe invention.
  • the term modulate includes effects on ABC transporters that enhances amyloid deposition, e.g., increase the production of amyloid in an animal model used to screen drugs for their ability to reduce amyloid deposition.
  • the blocker can affect an ABC transporter's ability to transport A ⁇ extracellularly from a cell, modulate the cleavage of amyloid precursor protein (APP), and modulate the proteolytic processing of APP.
  • the ABC transporter blocker is one or more lipophilic agents.
  • the blocker acts as a substrate for one or more ABC transporters.
  • altered expression includes effects upon the level of expression of either the mRNA or protein which encodes an ABC transporter.
  • Representative blockers useful in the present invention include, but are not limited to verapamil, desmethoxyverapamil, chloroquine, quinine, chinchonidine, primaquine, tamoxifen, dihydrocyclosporin, yohimbine, corynanthine.
  • blockers useful in the present invention include RU- 49953, verapamil, desmethoxyverapamil, cyclosporin, chloroquine, quinine, chinchonidine, primaquine, FK-506, MS-209, MS-073, S 9788, AHC-52, tamoxifen, dihydrocyclosporin, yohimbine, corynanthine, reserpine, physostigmine, acridine, acridine orange, quinacrine, chlo ⁇ romazine, propanolol, atropine, tryptamine, forskolin, 1, 9-dideoxyforskolin, PSC-833, VX-710, VX-853, GF120918, XR-9051 and trifluoperazine.
  • blockers useful in the present invention include RU-486, RU-49953, verapamil, desmethoxyverapamil, cyclosporin, chloroquine, quinine, chinchonidine, primaquine, FK-506, MS-209, MS-073, S 9788, AHC-52, tamoxifen, dihydrocyclosporin, yohimbine, corynanthine, reserpine, physostigmine, acridine, acridine orange, quinacrine, chlorpromazine, propanolol, atropine, tryptamine, forskolin, 1, 9-dideoxyforskolin, PSC-833, VX-710, VX-853, GF120918, XR-9051 and trifluoperazine.
  • the ABC transporter blocker is not RU-486.
  • Preferred blockers include PSC 833, MS-209, VX-710, VX-853, GF 120918, XR-9051, S 9788 and RU-49953.
  • RU-486 is generally associated with steroid hormone receptors such as glucocorticoid and progesterone receptors.
  • RU-486 acts as an antagonist toward these receptors and most commonly is used to block their ability to alter gene transcription (Moguilewsky M. and Philbert D., RU38486: "Potent anti-glucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by impaired activation.” J. Steroid Biochem. 20:271-276(1984); Meyer et al. "Agonistic and antagonistic activities of RU-486 on the functions ofthe human progesterone receptor.” EMBO J. 9:3923-3932 (1990)).
  • Steproid hormone receptor agonists and antagonists such as RU-486 are also able to act in a "nongenomic” fashion, altering tyrosine phosphorylation, neurotransmitter release, the concentration of intracellular calcium, inositol triphosphate production, levels of intracellular cAMP, and membrane potential.” (Wehling, M. "Nongenomic actions of steroid hormones", Trends Endocrinol. Metab. 5:347-353, (1994)). RU-486 also causes dissociation ofthe hetero- oligomeric complex of proteins to which unliganded steroid hormone receptors are bound resulting in release and activation of steroid hormone receptor associated proteins (Lebeau, M. C.
  • Steroid antagonists and receptor-associated proteins Human Reproduction 9(Suppl)2:l 1-21 (1994)
  • Steroid hormone receptors and structurally related antagonists also are known to act upon numerous membrane proteins, in particular neurotransmitter receptors (Brann D. W. et al. Emerging diversities in the mechanism of action of steroid hormones, J. Steroid Biochem. Mol. Biol. 52:113-133, (1995)).
  • RU49953 (17b-hydroxy-l lb, 17a-[4-dimethylaminophenyl]- 17a prop- 1-ynyl estra-4, 9-dien-3 one) is a derivative ofthe antiglucocorticoid/antiprogestin RU486 (17b- hydroxy-1 lb-[4-dimethylaminophenyl]-17a prop- 1-ynyl estra-4, 9-dien-3 one) which does not bind to either glucocorticoid or progesterone receptors to any appreciable degree (Marsaud V, Mercier-Bodard C, LeBihan S, Renoir JM, "Dexamethasone and triamcinoloneacetonide uptake by mouse fibroblasts is differently modulated by the immunosuppressants cyclosporin A, FK506, rapamycin and their analogues, as well as by other p-glycoprotein ligands", J Steroid Bio
  • disease state is intended to include those diseases, disorders or conditions which are associated with an increased amount of amyloid deposition, relative to a subject not afflicted with the disease, disorder or condition, in that the deposition of amyloid is directly or indirectly a causative agent ofthe disease, disorder or condition.
  • the amyloid deposition does not have to be the sole causative agent ofthe disease, disorder or condition but be merely responsible for causing some ofthe symptoms typically associated with the disease, disorder, or condition being treated.
  • Amyloid deposition can be the causative agent alone or at least one other agent can be involved in the state being treated.
  • Alzheimer's Disease head trauma, stroke, e.g., cerebral ischemia, Down's syndrome, hereditary cerebral hemorrhage amyloidosis, familial Mediterranean Fever, familial amyloid nephropathy with urticaria and deafness [Muckle- Wells syndrome], myeloma or macroglobulinemia, chronic hemodialysis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy, adult onset diabetes, insulinoma, gelsolin, cystatin C (hereditary cerebral hemorrhage with amyloidosis), familial amyloidotic polyneuropathy, Scrapie, Creutzfeldt- Jacob disease, kuru. Gerstmann-Straussler-Scheinker syndrome, bovine spongiform encephalopathy.
  • Preferred examples include those symptoms associated with Alzheimer's Disease, stroke and head trauma
  • the present invention provides methods for treating head trauma by administering to a subject having head trauma an effective amount of at least one ABC transporter or flippase blocker, or a pharmaceutically acceptable salt thereof, such that treatment occurs.
  • the present also provides methods for treating stroke by administering to a subject affected by a stroke an effective amount of at least one ABC transporter or flippase blocker, or a pharmaceutically acceptable salt thereof, such that treatment occurs.
  • lipophilic agent refers to a compound, such as a therapeutic agent, which, as a separate entity, is more soluble in nonpolar solvents than water.
  • a therapeutic agent which, as a separate entity, is more soluble in nonpolar solvents than water.
  • verapamil is considered lipophilic because it has greater solubility in hexane than in water.
  • the lipophilic nature of a lipophilic agent is related to its structure.
  • the agent can include lipophilic substituents such as saturated or unsaturated, substituted or unsubstituted alkyl, aryl or heteroaryl groups.
  • Such groups include substituted and unsubstituted, normal, branched or cyclic alkyl groups having 3 or more carbon atoms, substituted or unsubstituted arylalkyl or heteroarylalkyl groups and substituted or unsubstituted aryl or heteroaryl groups.
  • the lipophilic nature ofthe therapeutic agent can also be attributed to the "backbone" ofthe compound.
  • the backbone ofthe compound is that portion ofthe structure to which substituents are attached and can include lipophilic groups such as steroidal groups, saturated or unsaturated, substituted or unsubstituted alkyl, aryl or heteroaryl groups, or other fused cyclic ring systems.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci -C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer.
  • cycloalkyls have from 4-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • alkyl as used throughout the specification and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons ofthe hydrocarbon backbone.
  • substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxy alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxy carbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, tri
  • aryl as used herein includes 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles", “heteroaryls” or “heteroaromatics”.
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino.
  • substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbon
  • Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Preferred alkyl groups are lower alkyls having one to three carbon atoms.
  • heterocyclyl or “heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 4- to 7-membered rings, which ring structures include one to four heteroatoms.
  • Heterocyclyl groups include pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, mo ⁇ holine, lactones, lactams such as azetidinones and pyrrolidinones, lactones, sultams, sultones, and the like.
  • the heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamo
  • cycloalkyls e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls
  • rings e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls
  • bridged rings Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings ofthe polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
  • substrate means the specific compound acted upon by an enzyme, transporter, or other cellular proteins.
  • modulation of amyloid deposition means that amyloid deposition is prevented or decreased, e.g. AJ3 deposition. This modulation can be by one or more chemically induced physiological mechanisms.
  • the blocking agents and/or the lipophilic agents ofthe present invention can modulate amyloidosis in a subject such as by acting as an ATP binding cassette (ABC) transporter blocker, more preferably as a blocker of an ABC transporter expressed in the brain or cerebral microvasculature as described supra.
  • the blockers and/or the lipophilic agents ofthe present invention can modulate the cleavage of amyloid precursor protein (APP).
  • the blocker and/or lipophilic agent can modulate proteolytic processing of APP, thereby decreasing production of amyloid- ⁇ protein (A ⁇ ).
  • the blockers and/or the lipophilic agents ofthe present invention modulate proteolytic processing of APP, thereby increasing production of soluble amyloid precursor protein (APP S ).
  • the blockers and/or lipophilic agents ofthe present invention can modulate an ABC transporter's ability to export A ⁇ from a cell. Most preferably, the blockers and/or lipophilic agents ofthe present invention inhibit or prevent export of A ⁇ from a cell.
  • the term "modulate" is intended to mean that amyloid deposition is increased, e.g. A ⁇ deposition.
  • This modulation can be induced by one or more chemically induced physiological mechanisms.
  • effective amounts of a chemical agent can be screened for increased amyloid deposition in a subject.
  • the increase can be evaluated by comparing a subject treated with an agent ofthe present invention to a similar subject who was not treated.
  • the subject can have pre-existing amyloid deposits; therefore, the chemical agent serves to enhance the existing deposits.
  • the increase is at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to an untreated subject.
  • subject is intended to include mammals having amyloid deposition, including one or more amyloid related symptoms, or which are susceptible to amyloid deposition. Examples of such subjects include humans, dogs, cats, pigs, cows, horses, rats and mice.
  • administering is intended to include routes of administration which allow the ABC transporter blocker and/or the lipophilic agent to perform its intended function, e.g., preventing or inhibiting amyloidosis.
  • routes of administration include, but not necessarily limited to parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), oral (e.g., dietary), topical, nasal, rectal, or via slow releasing microcarriers depending on the disease or condition to be treated.
  • Oral, parenteral and intravenous administration are preferred modes of administration.
  • Formulation ofthe compound to be administered will vary according to the route of administration selected (e.g., solution, emulsion, gels, aerosols, capsule).
  • compositions comprising the compound to be administered can be prepared in a physiologically acceptable vehicle or carrier and optional adjuvants and preservatives.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media, sterile water, creams, ointments, lotions, oils, pastes and solid carriers.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. (1980)).
  • an effective amount is that amount ofthe blocker which allows it to perform its intended function.
  • an effective amount is that amount sufficient to inhibit, partially or totally, amyloid release, production and/or deposition or to reverse amyloid deposition or prevent or reduce its further deposition.
  • the "effective amount” also includes the amount sufficient to treat amyloidosis or Alzheimer's disease.
  • the effective amount will depend upon a number of factors, including biological activity ofthe blocker and/or lipophilic agent, age, body weight, sex, general health, severity of the disease to be treated, as well as appropriate pharmacokinetic properties.
  • dosages ofthe active substance can be from about 10 mg/kg/day to about 1000 mg/kg/day.
  • a therapeutically effective amount of the active substance can be administered by an appropriate route in a single dose or multiple doses. Further, the dosages ofthe active substance can be proportionally increased or decreased as indicated by the exigencies ofthe therapeutic or prophylactic situation.
  • amyloidosis is art recognized and is intended to include amyloid deposition related symptoms, such as progressive and undesirable memory impairment, loss of language and visuospatial skills, and behavior deficits. These changes in cognitive function are likely the result of degeneration of neurons in the cerebral cortex, hippocampus, basal forebrain, and other regions ofthe brain. The presence of large numbers of neurofibrillary tangles in degenerated neurons, neuritic plaques in the extracellular space and in the walls ofthe cerebral microvasculature are thought to be a result of amyloid deposition. For example, neuritic plaques consist of deposits of proteinaceous material surrounding an amyloid core.
  • pharmaceutically acceptable salt is intended to include pharmaceutically acceptable salts capable of being solvated under physiological conditions.
  • examples of such salts include sodium, disodium, potassium, dipotassium, and hemisulfate.
  • the term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g. alkyl esters, methyl, ethyl and propyl esters.
  • the present invention further pertains to packaged pharmaceutical compositions for treating amyloidosis.
  • the package includes a container for holding an effective amount of a pharmaceutical composition and instructions for using the pharmaceutical composition for treatment of amyloidosis.
  • the pharmaceutical composition includes at least one ABC transporter blocker for modulating amyloid deposition in a subject.
  • pharmaceutical composition includes blockers and/or lipophilic agents ofthe present invention and includes ingredients, such as other therapeutically active substances, inert ingredients, and carrier compounds.
  • the components ofthe composition must be compatible, meaning that the components must be capable of being commingled with the active substance, e. g.
  • the pharmaceutical compositions can be prepared by known procedures using well known and readily available ingredients.
  • the active substance will usually be admixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container.
  • the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or in a liquid medium), ointments containing up to 10% by weight ofthe active compound, soft and hard gelatin capsules, packaged powders, and the like.
  • Suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methylhydroxybenzoates, propylhydroxybenzoates, propylhydroxybenzoates, talc, and other compounds as are well known to those skilled in the pharmaceutical arts.
  • Blockers and/or lipophilic agents ofthe invention can be administered alone or in conjunction with other pharmacologically active agents, e.g., together with immunosuppressive agents or together with antibiotics and/or antiviral agents.
  • Compounds that can be coadministered include steroids (e.g. methyl prednisolone acetate), NSAIDs and other known immunosuppressants such as azathioprine, 15- deoxyspergualin, mizoribine, mycophenolate mofetil, brequinar sodium, leflunomide, and related molecules. Dosages of these drugs will also van' depending upon the condition and individual to be treated.
  • Blockers and/or lipophilic agents ofthe invention can be administered prior to the onset of amyloidosis, or after the onset of amyloidosis.
  • the blockers and/or lipophilic agents also can be administered as a prodrug which is converted to another form in vivo.
  • the present invention also pertains to methods for identifying agents which modulate amyloid production in an organism by administering to an organism, e.g., a transgenic mouse model which overexpresses A ⁇ , resulting in amyloid deposits, an effective amount of at least one ATP binding cassette (ABC) transporter blocker, such that modulation of amyloid deposition occurs.
  • ABSC ATP binding cassette
  • amyloid production, release or deposition occurs in membrane vesicles, cells, or organisms is used to identify the agents.
  • either the ABC transporter blocker or another agent induces a change in ABC transporter expression or stability which enhances amyloid production and can be used to produce a model for amyloidosis; in one embodiment, the model is an animal model, although other organisms, cell lines and even membranes may be useful in this regard. These models can be used to screen agents or drugs for their ability to reduce or prevent amyloid deposits.
  • organism is intended to include single cells, such as E. coli, multicellular organisms such as yeast and C. elegans cell lines and multicellular organisms including mammals, such as mice, rats, guinea pigs, and pigs that can develop amyloidosis.
  • multicellular organisms include transgenic animals, e.g., mammals, as well as those mammals identified as capable of developing amyloidosis, e.g., having amyloid deposition.
  • method for identifying agents includes assays and the like suitable for determining what agent or agents (blockers and/or lipophilic agents) elicit a response by the assay method.
  • combinatorial libraries can be screened to determine whether any members ofthe library have a desired activity, and, if so, to identify the active species. Methods of screening combinatorial libraries have been described (see, e.g., Gordon et al., J Med. Chem., op. cit). Soluble compound libraries can be screened for modulating transport of amyloid across cell membrane, followed by identification ofthe isolated compounds by conventional techniques (e.g., mass spectrometry, NMR, and the like).
  • the library compounds are conjugated to a label (e.g., fluorophores, colorimetric enzymes, radioisotopes, luminescent compounds, and the like) that can be detected to indicate reduction in ABC transport of amyloid.
  • a label e.g., fluorophores, colorimetric enzymes, radioisotopes, luminescent compounds, and the like
  • immobilized compounds can be selectively released and allowed to interact with an ABC transporter .
  • Exemplary assays useful for screening the libraries ofthe invention are known in the art (see, e.g., E. M. Gordon et al., J. Med. Chem. 37:1385-1401 (1994)).
  • cell lines can be developed which over-express each member ofthe family of ABC transporters, preferably, those ABC transporters expressed in the brain and/or the cerebral microvasculature, e.g., MDR1, MDR3, ABC1, ABC2, ABC3, ABC7, ABC8, MRP4, MRP5 and the human ABC transporters encoded by the ESTs 45597, 122234, 123147, 131042, 157481, 182763, 352188 or 422562, thereby providing surfaces of such cells with thousands of copies of a particular ABC transporter (For similar techniques see: Shapiro, A. B. and Ling, V., Reconstitution of Drug Transport by Purified P-Glycoprotein, Journal of Biological Chemistry, 270: 16167-16175, (1995)).
  • inside-out plasma vesicles can be constructed, thereby externalizing all ofthe molecular architecture found inside cells. Addition of ATP to these vesicles, causes the ABC transporter to move substrates across the cell membrane, except rather than moving substrates from inside (intracellular) to outside (extracellular), the substrate now becomes concentrated inside the vesicle. Reliable preparations of plasma vesicles that transport amyloid can be used to screen chemical libraries to isolate new compounds capable of reducing ABC transport of amyloid.
  • the present invention further pertains to in vitro and in vivo methods for identifying agents which modulate transport of amyloid across membranes, e.g., cellular or synthetic membranes.
  • the methods include introducing an agent into a model system which contains a membrane, an ABC transporter expressed in the brain or cerebral microvasculature and amyloid. The ability ofthe agent to modulate the transport of amyloid across the membrane is measured.
  • Suitable synthetic membranes are those composed of lipids as described in (Slatin, S. "Colicin E in planar lipid bilayers" Internat. J. Biochem.
  • the present invention also pertains to in vitro methods for inserting ABC transporters, preferably those ABC transporters expressed in the brain and/or cerebral microvasculature, e.g., MDR1, MDR3, ABC1, ABC2, ABC3, ABC7, ABC8, MRP4, MRP5 and the human ABC transporters encoded by the ESTs 45597, 122234, 123147, 131042, 157481, 182763, 352188 or 422562, into planar lipid bilayers as a means of assaying regulation of amyloid transport.
  • purified or recombinant proteins can be inserted into planar lipid bilayers (Slatin, S. "Colicin E in planar lipid bilayers" Internat. J. Biochem.
  • the methods include introducing an agent into a model system which contains a planar lipid bilayer, an ABC transporter and amyloid. The ability ofthe agent to modulate the transport of amyloid across the planar lipid bilayer can be measured.
  • model system includes cells, cell lines, mammals, birds, insects, single- and multicellular organisms and in vitro systems.
  • Immature APP is believed to be transported through intracellular secretory machinery to the membrane where it is found as a transmembrane protein known as mature APP.
  • APP can be cleaved by ⁇ - and ⁇ - secretases; the cleavage occurs at the N- and C-termini of A ⁇ (respectively), resulting in production of A ⁇ peptide.
  • mature APP undergoes one of several proteolytic processing routes.
  • the ⁇ -secretory processing of APP involves cleavage of the molecule in the extracellular domain, near the membrane and within the A ⁇ sequence.
  • This cleavage of APP has two potential consequences: (1) it precludes formation of A ⁇ ; and (2) solubilizes the extracellular domain of APP which is then released into the extracellular space.
  • An alternative route involves internalization to an endosomal compartment with potential for ⁇ - and ⁇ -secretase cleavage, once again resulting in production of A ⁇ .
  • ABC transporters export A ⁇ from cells.
  • MDR1, MDR3, ABC1, ABC2, ABC3, ABC7, ABC8, MRP4, MRP5 and the human ABC transporters encoded by the ESTs 45597, 122234, 123147, 131042, 157481, 182763, 352188 or 422562 are members ofthe ATP-binding cassette (ABC) superfamily of transporters; using ATP as an energy source, certain ABC transporters such as MDR1 are known to pump various molecules against their concentration gradients (Endicott, J. A. and V. Ling, "The biochemistry of P-glycoprotein-mediated multidrug resistance", Annu. Rev. Biochem. 58:137-171 (1989); Gottesman, M. M. and I. Pastan,
  • MDRl act as substrates for MDRl; while structurally unrelated, all share the property of being exceedingly hydrophobic (Zamora, J. M. et al, "Physical-chemical properties shared by compounds that modulate multidrug resistance in human leukemia cells", Mol. Pharmacol. 33:454-462 (1988), the teachings of which are hereby inco ⁇ orated by reference) which has led to the belief that MDRl act as either a "hydrophobic vacuum cleaner” for a wide variety of membrane- associated hydrophobic molecules or as a flippase (Higgins. C. F. and M. M.
  • a ⁇ is a small, lipophilic peptide, it has appropriate properties to act as a substrate for MDRl .
  • flippase is art recognized and is intended to include the ability of an ABC transporter to act as a flipping agent, e.g., to move A ⁇ from the inner leaflet of a lipid bilayer to the outer leaflet. That is, an ABC transporter acting as a flippase delivers A ⁇ to the outer leaflet ofthe lipid bilayer, in this scenario, other molecules may be involved in moving A ⁇ from the outer leaflet ofthe lipid bilayer into the extracellular space.
  • the flippase action ofthe ABC transporter is critical to the multi- factorial process leading to A ⁇ efflux.
  • flippase blocker is intended to include those compounds that can modulate ABC transporters expressed in the brain or the cerebral microvasculature, such as those described supra, including MDRl, MDR3, ABCl, ABC2, ABC3, ABC7, ABC8, MRP4, MRP5 and the human ABC transporters encoded by the ESTs 45597, 122234, 123147, 131042, 157481, 182763, 352188 or 422562.
  • ABC transporters indeed function as transporters in cells include the observation that the human MDR3 gene product promotes translocation of phosphatidylcholine (Smith et al., "The human MDR3 p- glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice” FEBS Lett 354:263-266 (1994)), that the ABC transporter known as abcl appears to be involved in secretion of interleukin 1 ⁇ (Hamon et al., "Interleukin- l ⁇ secretion is impaired by inhibitors of the ATP binding cassette transporter, ABCl " Blood 90:2911-2915 (1997)), and that the transport of certain peptides into the lumen ofthe endoplasmic reticulum is accomplished by the conjoint efforts of two members ofthe ABC transporter family known as TAP1 and TAP2 (Heemels MT and Ploegh H, "Generation, translocation, and presentation of MHC class
  • a ⁇ is released from all cells, and thus it is reasonable to assume that all cells have a mechanism for A ⁇ release.
  • MDRl MDRl
  • this ABC transporter is involved in the process of A ⁇ efflux.
  • neurons produce and release prodigious amounts of A ⁇ (Busciglio et al., "Generation of ⁇ -amyloid in the secretory pathway in neuronal and nonneuronal cells," Proc Natl Acad Sci USA 90:2092-2096 (1993); Simons et al., "Amyloidogenic processing ofthe human amyloid precursor protein in primary cultures of rat hippocampal neurons," J Neurosci 16:899- 908 (1996)).
  • ABC transporters are involved in the process of A ⁇ efflux in brain.
  • amyloid deposition in Alzheimer's disease occurs both in the parenchyma ofthe brain and in the microvasculature (Selkoe, DJ “The molecular pathology of Alzheimer's disease,” Neuron 6:487-498 (1991))
  • ABC transporters expressed in brain and the microvasculature represent key targets for regulating A ⁇ release via the present invention.
  • brain-and microvasculature- expressing ABC transporters represent preferred targets for the development of Alzheimer's disease therapeutics.
  • ABC transporters MDRl, MDR3, ABCl, ABC2, ABC3, ABC7, ABC8, MRP4, MRP5 and the human ABC transporters encoded by the ESTs 45597, 122234, 123147, 131042, 157481, 182763, 352188 or 422562 can function to allosterically modify the function of other membrane proteins.
  • modulation of p-glycoprotein by ABC transporter blockers has been shown to alter the magnitude of volume-activated chloride currents (reviewed in Higgins, C. F. Volume- activated chloride currents associated with the multidrug resistance P-glycoprotein, J. Physiol. 482:31S-36S (1995)).
  • p-glycoprotein and other ABC transporters have multiple functions, one of which is to allosterically modify the function ofthe other membrane proteins.
  • the present invention is consistent with a model in which allosteric modification of other membrane proteins by an ABC transporter is responsible for the change in APP catabolism. such that A ⁇ release is reduced, and/or in which the ABC transporter exports A ⁇ . From this, it follows that pharmacological agents which alter the ability of these molecules to export A ⁇ or the expression of such molecules can be therapeutically useful in Alzheimer's disease.
  • Murine nerve growth factor was purchased from GIBCO and dexamethasone (DEX) was purchased from SIGMA.
  • Mifepristone (RU-486) was purchased from Research Biochemicals International (RBI, Natick, MA) through the NIMH (National Institute for Mental Health) Chemical Synthesis Program.
  • Cyclosporin A (CsA) was purchased from RBI.
  • the Anti-Alzheimer Precursor Protein A4 (22C11) monoclonal antibody specific to the pre-A4 molecule (amyloid precursor protein) was purchased from Boehringer Mannheim.
  • the GSrasDNl PC 12 subline (Dr. Simon Halegous (State University of New York at Stony Brook)) expressing the dominant inhibitory mutant ras gene, p21 ⁇ 17 under the control of a dexamethasone-inducible MMTV promoter (mouse mammary tumor virus), obtained as a gift from Dr. Simon Halegoua, (Kremer et al, J. Cell Biol. 115(3):809-819 (1991)) and grown in Dulbecco's modified Eagles' medium (DMEM, GIBCO) containing 5% fetal bovine serum (FBS: v/v) and 10% heat inactivated horse serum (HS, GIBCO).
  • DMEM Dulbecco's modified Eagles' medium
  • FBS v/v
  • H heat inactivated horse serum
  • RU-486 and NGF were solubilized in dimethylsulfoxide (DMSO).
  • DMSO dimethylsulfoxide
  • GSrasDNl PC 12 cells For drug exposure experiments in GSrasDNl PC 12 cells, cells were grown to confluence in 75 cm polypropylene culture flasks from Falcon. Twelve hours prior to drug treatment, the culture medium was aspirated using a pasteur pipette and replaced with 10 ml of DMEM supplemented with 15% charcoal-stripped calf serum (SIGMA) to remove complement, immunoglobulins and endogenous steroid hormones secreted from the cells. Twelve hours later, this supplemental medium was aspirated and replaced with 10 ml of DMEM containing the treatment drugs. Flasks were allotted into the following treatment groups: a) Control and b) RU-486 (3.0 ⁇ M). DMSO concentrations were normalized to 0.06% (v/v) in all flasks.
  • SIGMA charcoal-stripped calf serum
  • the resultant cell pellet was resuspended first using a 20-1/2 gauge needle followed by a 22-1/2 gauge needle. 8 ml of DMEM was added to make a final volume of 10 ml. 13 ⁇ l of this cell suspension was taken for cell counting using a haemocytometer. A dilution was performed using DMEM to make a final cell suspension with a density of 1.0 x 10" cells/ml. 10 mis of cells from each treatment group was pipetted into 15 ml conical tubes labeled CONTROL, NGF, or RU-486. Drugs were reintroduced to the cells at the same concentration as described above for the 12-hour pretreatment.
  • NGF 50 ng/ml was added to the tube of cells labeled 'NGF' as a positive control.
  • 1.0 ml of cells from each group was pipetted into appropriately labeled 1.5 ml eppendorf tubes (BIORAD) and placed into a 5% CO2 incubator equilibrated at 37°C for fifteen minutes. After the fifteen minutes had elapsed, the tubes were placed immediately on ice to stop any reactions and 200 ⁇ l of a protease cocktail (100 ⁇ M PMSF (phenylmethanesulfonyl fluoride), 5 ⁇ g/ml Leupeptin, 5 ⁇ g/ml Aprotinin, and 5 ⁇ g/ml Pepstatin) was added to each tube.
  • PMSF phenylmethanesulfonyl fluoride
  • Cells were pelletted at 14,000 ⁇ m for five minutes at 4°C and the medium was removed and collected into fresh, ice-cold 1.5 ml eppendorf tubes containing protease inhibitor cocktail. Cell pellets were lysed using 50 ⁇ l of met/lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Nonindet P40 (v/v), and 1% Na deoxycholate (w/v), pH 7.4). The nuclear fraction was pelletted down at 14,000 ⁇ m for ten min. and 5 ⁇ l of cellular homogenate was collected for a protein quantification.
  • met/lysis buffer 10 mM Tris-HCl, 150 mM NaCl, 1% Nonindet P40 (v/v), and 1% Na deoxycholate (w/v), pH 7.4
  • APP s -containing medium was desalted using ultrafree-CL centrifugation filters with a 30,000 MW cut-off range membrane (Millipore). The desalted supernatant was placed in 1.5 ml eppendorf tubes and concentrated using a speed vac-concentrator. 30 ⁇ l of Laemmli sample buffer (0.0625 Tris-HCL, 2% SDS, 10% glycerol, 5% 2- mercaptoethanol, 0.002% bromophenol blue) was added to reconstitute the pellet. A bicinchoninic acid (BCA) protein assay kit from Pierce (PO Box 117,
  • RU-486 was used as a negative control group for the expression ofthe glucocorticoid-inducible MMTV promoter-linked dominant negative ras gene product in these cells. In these experiments, the effects of prolonged exposure of RU-486 (twelve hours) on these cells were studied and APP S was collected for fifteen minutes.
  • NGF is a known enhancer of APP S secretion in PC 12 cells (Fukuyama, R. et al, "Nerve growth factor-induced neuronal differentiation is accompanied by differential induction and localization ofthe amyloid precursor protein (APP) in PC 12 cells and variant PC12S cells", Mol. Brain Res. 17:17-22 (1993); Haring, R.
  • Phorbol 12-myristate 13-acetate was purchased from LC Service Co ⁇ ., Woburn, Mass. All other materials were obtained as previously described in Example 1. Wild-type PC 12 cells were purchased from the American Type Tissue Culture Collection # CRL-1721 and grown in DMEM with 5% FBS fetal bovine serum and 10% horse serum (GIBCO).
  • PC 12 cells grown to confluence were pretreated for twelve hours in DMEM supplemented with charcoal-stripped calf serum (SIGMA) as described above. After pretreatment, cells were trypsinized, resuspended, and counted as described above. 10 mis each of 1.0 x 10° cells/ml PC 12 cells were aliquoted into 15 ml conical tubes containing the appropriate treatment group. PMA was used as a positive control as previous experiments had shown that such treatment increases APP S release (Buxbaum, J. D. et al, "Processing of Alzheimer ⁇ /A4 amyloid precursor protein: Modulation by agents that regulate protein phosphorylation", Proc. Natl. Acad. Sci.
  • RU-486 increased APP S secretion in wild-type PC 12 cells following fifteen minute exposure (Fig. 5). The data indicated that the effect previously shown was not due to an effect caused by the presence ofthe dominant-negative ras gene in the
  • GSrasDNl PC12 cell line Since it is generally accepted that genomic effects of steroid hormone receptor activation require at least one hour to manifest (Wehling, M, Trends Endocrinol Metab. 56:347-353, 1994), the data demonstrate that a twelve hour pretreatment with RU-486 was not necessary for an RU-486-mediated increase in APP S secretion. The data also demonstrated that APP S secretion was not dependent upon a change in gene transcription.
  • Fig. 5 depicts a western blot which shows varying intensities in APP S isoforms, most likely due to variations in post-translational processing of isoforms. Since each band shows corresponding changes upon treatment with RU-486, it can be concluded that RU-486 affects the release of all isoforms of APP S , although subtle isoform selective effects have not been ruled out.
  • the increase in APP S secretion with PMA confirms published findings (Buxbaum, J. D. et al, "Processing of Alzheimer ⁇ /A4 amyloid precursor protein: Modulation by agents that regulate protein phosphorylation", Proc. Natl. Acad. Sci. USA 87:6003-6006 (1990); Caporaso, G. L.
  • E82 mouse L-cells were generously provided by Dr. Mark Danielson (Georgetown University, USA). All materials and methods are as described above. E82 cells were grown in DMEM with 5% FBS and cultured in 75 cm2 flasks from Falcon.
  • RU-486 increases APP S release from E82 cells. These cells (Housley, P. R. and Forsthoefel, A. M., Isolation and characterization of a mouse L cell variant deficient in glucocorticoid receptors, Biochem. Biophys. Res. Comm. 164:480-487, 1989)) lack steroid hormone receptors with which RU-486 is known to interact, e.g., the glucocorticoid and progesterone receptors (Baulieu, Science 245:1351-1357, 1989). The increase in APP S secretion using RU-486 in these cells (Fig. 6) further demonstrates that the observed effects occurs independent of RU-486 activity at steroid hormone receptors.
  • Dexamethasone was purchased from Sigma.
  • Progesterone (HBC complex) and desmethoxyverapamil were purchased from Research Biochemicals Inc.
  • Treatment groups were as follows: a) Control; b) PMA (0.1 ⁇ M); c) RU-486 (0.1 ⁇ M); d) dexamethasone (0.1 ⁇ M); progesterone (1 ⁇ M); desmethoxyverapamil (1 ⁇ M).
  • a KB-3.1 human carcinoma cell line expressing low levels ofthe human MDRl gene encoding P-glycoprotein gene product (Shen et al, 1986, Science, 232; 643-5), was obtained as a gift from Dr. Ira Pastan (National Cancer Institute, USA). These cells were grown in DMEM with 10%> FBS. All materials and methods are as outlined as above.
  • KB-3.1 cells expressing sub-detectable levels of p-glycoprotein lack modulation of APP S by RU-486 (Fig. 9).
  • This experiment directly addresses the involvement of p- glycoprotein in regulating APP catabolism.
  • the observation that cells having low expression of p-glycoprotein do not regulate APP S secretion either by RU-486 or PMA (Fig. 9) suggests that the catabolism of APP may be linked in some fashion to the level of expression of p-glycoprotein.
  • Western blots were performed on PC 12, E82, and KB- 3.1 cells probing for cellular levels of p-glycoprotein. Detectable levels ofthe protein were found in PC 12 and E82 cells, but none in KB-3.1 cells (data not shown).
  • this experiment is not conclusive nor definitive in implicating p-glycoprotein as the only molecule underlying RU-486 mediated APP S increase, it does provide direct support for this belief.
  • the cDNA was resuspended with 450 ⁇ l of 0.1 x tris-EDTA(TE) ethylene diamine tetraacetic acid, tris EDTA and 50 ⁇ l of 2.5 M CaCl 2 .
  • DMEM fetal calf serum
  • ⁇ -galactosidase ( ⁇ -gal) gene was also co-transfected into the cells, ⁇ -gal staining was performed to determine the percentage of cells within each dish which were successfully transfected with the cDNA. Exposures were performed without resuspending the cells, in contrast to previous experiments described above. 10 ml solutions of DMEM containing either control (DMSO), PMA(0.1 ⁇ M) or RU-486 (0.1 ⁇ M) were prepared. DMSO concentrations in the medium were normalized to 0.01%(v/v).
  • the supplemented medium was aspirated and 1 ml ofthe appropriate medium was added to the cells. Dishes were returned to the incubator for fifteen minutes and the medium harvested and processed as previously described.
  • the plated cells were lysed using 100 ⁇ l of met/lysis buffer and harvested with cell scrapers (Falcon). 5 ⁇ l from each sample was collected for protein quantification. Protein determination and APP S quantification were performed as previously described.
  • Figure 11 demonstrates that RU486, a P-gp blocker reduces A ⁇ secretion in fifteen minutes in a dose-dependent manner in K269sw cells transiently transfected with the human pHaMDRl construct.
  • K269sw cells were cultured in 20 mm Falcon dishes in DMEM containing 10% FBS, and 200 mg/mL geneticin (GIBCO) to about 50% confluence and transiently transfected with the human pHaMDRl construct using the calcium-phosphate precipitation technique as described above. Cells were replated at a density of 2.0 x 10 cells/dish 8 hours post-transfection on 10 cm petri dishes as described above.
  • ⁇ -gal staining was performed to determine the percentage of cells within each dish which were successfully transfected with the cDNA. Exposures were performed without resuspending the cells. 10 mL of DMEM containing either control (DMSO), PMA (0.1 ⁇ M), or RU486 (0.001 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M), were prepared. DMSO concentrations in the medium were normalized to 0.01%) (v/v). The supplemented medium was aspirated and 1 mL ofthe appropriate medium was added to the cells. Dishes were returned to the incubator for fifteen minutes and the medium harvested and processed as previously described. The plated cells were lysed using 100 ⁇ L of met lysis buffer and harvested with cell scrapers (Falcon). 5 ⁇ L from each sample was collected for protein quantification.
  • DMSO control
  • PMA 0.1 ⁇ M
  • RU486 0.001 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 1.0 ⁇
  • TCA trichloroacetic acid
  • Figure 12 demonstrates that RU49953, a selective P-gp blocker, reduces A ⁇ secretion in fifteen minutes in a dose-dependent manner in K269sw cells transiently transfected with the human pHaMDRl construct. Culturing and transient transfection of K269sw cells are as previously described. Preparation of cells for the drug assays are also performed as previously described.
  • Exposures were performed without resuspending the cells. 10 mL of DMEM containing either control (DMSO), PMA (0.1 ⁇ M), or RU49953 (0.01 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M), were prepared. DMSO concentrations in the medium were normalized to 0.01%> (v/v). The supplemented medium was aspirated and 1 mL ofthe appropriate medium was added to the cells. Dishes were returned to the incubator for fifteen minutes and the medium harvested and processed as previously described. The plated cells were lysed using 100 ⁇ L of met/lysis buffer and harvested with cell scrapers (Falcon). 5 ⁇ L from each sample was collected for protein quantification.
  • TCA trichloroacetic acid
  • FIG. 13 and 14 demonstrates that transient transfection of P-glycoprotein in K269sw cells increases basal secretion of ⁇ -amyloid.
  • K269sw cells were grown as previously described. Protocols for transient transfection are also as described albeit the following modifications. Individual 20 mm plates of K269sw cells were transfected with the following: control (no calcium-phosphate precipitation); mock (calcium- phosphate precipitation without any plasmid); ⁇ -gal (transfection with the ⁇ - galactosidase gene); and MDRl (transfection with the human MDRl gene). Replating of cells post-transfection was performed as described above.
  • Basal levels of A ⁇ secretion were assayed in the following way. Supplemented medium was replaced with 1 mL DMEM and dishes were returned to the incubator for one hour. Conditioned medium was harvested and secreted A ⁇ was detected and quantified as previously described.
  • XI Exemplary in vitro screening assay for identifying agents which modulate transport of A ⁇ across membranes
  • An exemplary in vitro screening assay for identifying agents which modulate transport of A ⁇ across membranes by measuring A ⁇ transport across the membrane is set forth below.
  • Liposome transport of A ⁇ Liposomes derived from AuxBl (p-gp deficient) and CHRB30 (p-gp enriched) cell lines were incubated at 37°C for 15 minutes with lOOnM A ⁇ 1-40/1 -452 (US Peptides Inc.) to allow for peptide association with the membrane. Excess A ⁇ was removed by passing the liposomes through a BioGel P-6 size exclusion column (BioRad). A final concentration of 1.5 mM Na4ATP (Sigma) or AMP-PNP (Sigma) was added to the liposomes to activate p-gp and the entire preparation was left to react for 15 minutes at 37°C.
  • western blots using the 6E10 antibody show levels of membrane-bound A ⁇ peptides (A ⁇ MEMB) and their corresponding levels in the interior of the liposome (A ⁇ lNTRA) before and after addition of nucleotide.
  • Fig. 15D shows overexposed a western blot of synthetic A ⁇ peptides spun through a Biogel-P6 size exclusion column (BG-P) compared to standards (Std). 100 nM A ⁇ standards develop an intense signal while eluant collected from solution containing 100 nM A ⁇ spun through BioGel-P6 columns show no detectable signal even after overexposure ofthe blot to ECL film.
  • BG-P Biogel-P6 size exclusion column
  • Van Veen, H.W. et al. A bacterial antibiotic-resistance gene that complements the human multidrug-resistance P-glycoprotein gene. Nature 391, 291-295 (1998).

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Abstract

L'invention concerne des procédés de modulation de l'amylose chez un sujet. Une dose efficace d'au moins un bloqueur de transporteur de cassette de fixation ATP (ABC) est administrée à un sujet, de sorte que la modulation de l'amylose soit induite. Des procédés consistent également à administrer à un sujet une dose efficace d'au moins un bloqueur de transporteur d'ABC, ou un sel de celui-ci, acceptable au plan pharmaceutique, de manière que l'état pathologique associé à l'amylose soit traité. Des compostions pharmaceutiques conditionnées pour le traitement de l'amylose sont également décrites. Le conditionnement est constitué d'un récipient destiné à contenir une quantité efficace d'une composition pharmaceutique et d'instructions d'utilisation de la composition pharmaceutique pour le traitement de l'amylose. La composition pharmaceutique comprend au moins un bloqueur ABC pour la modulation de l'amylose chez un sujet. Des procédés d'identification d'agents qui modulent l'amylose chez un sujet sont également décrits. Une quantité efficace d'au moins un bloqueur de transporteur de cassette de fixation ATP (ABC) est administrée à un organisme, de sorte que la modulation de l'amylose soit induite.
PCT/US1999/023885 1998-10-23 1999-10-14 Procede et composition pour la modulation de l'amylose WO2000024390A1 (fr)

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JP2000578000A JP2002528411A (ja) 1998-10-23 1999-10-14 アミロイド症を変調する方法及び組成物
CA002348019A CA2348019A1 (fr) 1998-10-23 1999-10-14 Procede et composition pour la modulation de l'amylose
IL14272599A IL142725A0 (en) 1998-10-23 1999-10-14 Method and composition for modulating amyloidosis
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WO2002064781A2 (fr) * 2001-02-09 2002-08-22 Active Pass Pharmaceuticals, Inc. Regulation de l'expression de la proteine precurseur amyloide par modification de l'expression ou de l'activite du transporteur abc
WO2002070690A2 (fr) * 2001-03-02 2002-09-12 Active Pass Pharmaceuticals, Inc. Nouveaux transporteurs abca5 et leurs utilisations
US6617122B1 (en) 1999-03-15 2003-09-09 Xenon Genetics, Inc. Process for identifying modulators of ABC1 activity
WO2004047854A2 (fr) * 2002-11-22 2004-06-10 Novartis Ag Procedes de traitement de la maladie d'alzheimer et compositions afferentes
WO2004067550A2 (fr) * 2003-01-22 2004-08-12 Centre National De La Recherche Scientifique Nouvelle utilisation de la mifepristone et de ses derives comme modulateurs de la voie de signalisation des proteines hedgehog et ses applications
EP1470818A1 (fr) * 2003-04-25 2004-10-27 Neuro3D Utilisation de dérivés de phénothiazine pipérazine pour la préparation d'un médicament ayant des effets neuroprotecteurs et/ou neurotrophiques sur le SNC et/ou SNP
WO2006018850A3 (fr) * 2004-08-19 2006-08-10 L P Tel Aviv University Future Compositions et leurs methodes d'utilisation dans le traitement de maladies associees aux amyloides
WO2008039200A1 (fr) * 2006-09-27 2008-04-03 Irm Llc Procédés et compositions pour le traitement du lymphome et du myélome
WO2008122666A1 (fr) * 2007-04-10 2008-10-16 Inserm (Institut National De La Sante Et De La Recherche Medicale) Inhibiteurs de mrp4 utilises dans le traitement des troubles vasculaires
US7491699B2 (en) 2002-12-09 2009-02-17 Ramot At Tel Aviv University Ltd. Peptide nanostructures and methods of generating and using the same
US7504383B2 (en) 2003-01-07 2009-03-17 Ramot At Tel Aviv University Ltd. Peptide nanostructures encapsulating a foreign material and method of manufacturing same
WO2012159674A1 (fr) * 2011-05-26 2012-11-29 Universita' Degli Studi Di Bari Procédé de criblage pour détecter des composés thérapeutiques utiles pour la détoxification du système nerveux central en peptide b-amyloïde
US8372880B2 (en) 2003-09-25 2013-02-12 Tel Aviv University Future Technology Development L.P. Compositions and methods using same for treating amyloid-associated diseases
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